US 7545500 B2
The present invention discloses surface plasmon resonance phenomenon measuring equipment comprising:
1. Surface plasmon resonance phenomenon measuring equipment comprising:
(1) a prism,
(2) a sensor wherein a plurality of measuring cells are formed in m rows (m≧2) and n columns (n≧2) on a metal film formed on the bottom face of the prism,
(3) a light source for radiating a laser beam,
(4) a first optical system wherein m optical units each having a rectangular parallelepiped shape and having a translucent film formed along the diagonal surface of the rectangular parallelepiped are arranged continuously in the direction of the laser beam radiated from the light source and thereby the laser beam is converted into a transmitted light and a m parallel reflected lights A group,
(5) a second optical system fitted to one side of the prism, wherein optical units same as said m optical units are arranged in n columns continuously along the radiation directions of the m parallel reflected lights of said parallel reflected lights A group and thereby the parallel reflected lights A group is converted into transmitted lights and a mn parallel reflected lights group B, and wherein the reflected lights group B is radiated toward the measuring cells on the metal film formed on the bottom face of the prism, so as to hit the metal film at an incident angle including the plasmon resonance angle and thereby a reflected lights C group is radiated from other side of the prism,
(6) a photodiode array detectors group of m rows and n columns, arranged on the extensions of the reflected lights C group, and
(7) a polarizer interposed between a beam splitter comprising the first optical system and the second optical system and the prism, and/or between the prism and the photodiode array detectors group.
2. Surface plasmon resonance phenomenon measuring equipment according to
3. Surface plasmon resonance phenomenon measuring equipment according to
4. An optical apparatus for surface plasmon resonance phenomenon measuring equipment, which comprises:
a beam splitter having:
a first optical system wherein m optical units each having a rectangular parallelepiped shape and having a translucent film formed along the diagonal surface of the rectangular parallelepiped are arranged continuously in the direction of the laser beam radiated from the light source and thereby the laser beam is converted into a transmitted light and a m parallel reflected lights A group, and
a second optical system wherein optical units same as said m optical units are arranged in n columns continuously along the radiation directions of the m reflected lights of said parallel radiated lights A group and thereby the parallel reflected lights are converted into transmitted lights and mn parallel reflected lights B group,
photodiode array detectors of m rows and n columns, and
a connecting member for connecting the beam splitter and the photodiode array,
wherein the connecting member has a means for changing the angle and distance formed by and between the beam splitter and the photodiode.
The present invention relates to multi-channel surface plasmon resonance phenomenon measuring equipment wherein a plasmon resonance phenomenon is utilized as a principle of measurement and simultaneous analysis of a plurality of samples can be made.
Surface plasmon resonance phenomenon measuring equipment is an apparatus which enables the monitoring of food safety or environment or the high-sensitivity detection of dangerous articles or drugs. This equipment is expected to find applications in a number of fields such as environmental protection, medical care, agriculture, stock raising, food industry and the like.
Surface plasmon resonance (SPR) measuring equipment has been marketed by BIACORE AB, NIPPON LASER & ELECTRONICS LAB, etc. With these equipment, the number of samples measured at one time is one, making low the efficiency of measurement.
In order to make small the SPR measuring equipment to enable on-site measurement, the present inventors developed portable SPR sensors (Japanese Patent No. 3462179, No. 3335621 and No. 3356212) wherein a light radiated from a light source is passed through a cylindrical lens to form a liner focus, the light of linear focus is allowed to be incident on a sensor made of a prism and a glass substrate, and the reflected light from the prism is measured by a CCD linear sensor. Even with these measuring equipment, the number of samples measured at one time is one as well, making low the efficiency of measurement.
In order to increase the efficiency of measurement, there is required a function enabling simultaneous analysis of a large number of samples, i.e. a function of simultaneous measurement of multi-sample or multi-channeling.
There are two proposals for realizing the multi-channeling. The first proposal is an approach in which a light from a light source is divided into two light paths by a beam splitter, they are allowed to hit the pre-determined two points of a SPR sensor constituted by a prism, the attenuated lights caused by surface plasma resonance phenomenon are detected by two independent light detectors, then the detection signals are each amplified (Japanese Patent No. 3462179). The second proposal is an approach in which a reflected light from a prism is divided into two light paths by a light-splitting mirror and they are detected by respective light detectors (Japanese Patent Application No. 2003-118565).
With the multi-channeling by these approaches, however, the division into light paths is one-dimensional (linear) and the number of samples measured at one time is limited to about two. Therefore, a striking increase in the efficiency of measurement is impossible.
Hence, it is desired to develop surface plasmon resonance phenomenon measuring equipment which has a function of efficiently analyzing a large number of samples (e.g. four or more samples) at one time, i.e. a function of simultaneous measurement of multi-sample or multi-channeling.
The present inventors made a study in order to solve the above-mentioned problems. As a result, the present inventors devised a beam splitter for converting a laser beam as a light source into a large number of parallel lights and, by utilizing the beam splitter, enabled simultaneous measurement of multi-sample.
The present invention has been completed based on the above finding. The present invention aims at providing surface plasmon resonance phenomenon measuring equipment which utilizes a surface plasmon resonance phenomenon (SPR) and achieves multi-channel measurement of multi-sample.
The present invention, which has achieved the above aim, is described below.
In the present invention, a laser beam is divided into desired (m×n) laser beams by a beam splitter. As a result, the light paths of the reflected lights group B radiated onto measuring cells become two-dimensional (planar in m rows and n columns) and, correspondingly therewith, the photodiode array detectors group receiving the reflected lights radiated from the measuring cells become as well two-dimensional (planar in m rows and n columns). Consequently, the number of samples measured at one time can be increased strikingly, elevating the efficiency of measurement. Further, despite of simultaneous measurement of a number of samples, the size of measuring equipment can be made compact.
In the present invention, the intensities of lights after laser beam splitting by the beam splitter are different from each other; however, this difference can be corrected by conducting an operation at the amplifier section or the CPU. When there is provided, like this, an operation means for correction of the luminous energies of reflected lights C group in sample measurement, a more accurate measurement result can be obtained.
The optical system used in the present invention is superior in scalability, and all the optical elements from light source to prism and detector can be used by scaling-up or scaling-down. By scaling down the optical elements and combining them with micro-cells, a palm-sized SPR system can be manufactured; or, by scaling up, there can be manufactured a SPR system using a plate of 96 holes as measurement cells, which is equivalent to commercial SPR systems.
In the present invention, since no lens is used for convergence of light paths, the manufacturing cost of measuring equipment can be made low and the measuring equipment per se can be made compact.
In the measurement utilizing a surface plasmon resonance phenomenon, it is necessary to change the angle of SPR excitation light and the angle of SPR reflected light depending upon the intended application. This necessity can be satisfied by preparing a plurality of prisms different in angle and selecting an optimum prism from them so as to meet the intended purpose. Further, by providing a mechanism with which the beam splitter unit and the detector unit are automatically arranged and adhered so as to match the angle of prism, a prism can be fitted into an optical system by a simple operation.
100 is a light source; 110 is a laser beam; 200 is a beam splitter; 210 is a first optical system; 220 is a second optical system; 230 is a parallel reflected lights A group; 240 is a parallel reflected lights B group; 250 is a translucent film; 260 is a translucent film; 300 is a sensor; 310 is a glass plate; 320 is a metal film; 330 is a measuring cell; 340 is a reflected lights C group; 410 is a prism; 420 a is a polarizer; 420 b is a polarizer; 500 is a detector; 510 is a photodiode array detectors group; 710 is a rotation axis of beam splitter; 720 is a rotation axis of detector; 730 is a guide bar; 732 is a long hole; 810 is a glass plate; 820 is a translucent film; 830 is an adhesive layer; A is a splitting unit of first optical system; A1 to Am are a splitting units group of first optical system; B is a splitting unit of second optical system; B11 to B1 n . . . Bm1 to Bmn are a splitting units group of second optical system; LA1 is a reflected light from a splitting unit A1; LAm is a reflected light from a spitting unit Am; LB1 n is a reflected light from a splitting unit B1 n; LBm1 is a reflected light from a splitting unit Bm1; LBmn is a reflected lights group from a splitting units group BmN; θa is an incidence angle; and θb is a reflection angle.
The present invention is described in detail below with reference to the accompanying drawings.
The incidence angle θa of each reflected light on the sensor is an incidence angle including the plasmon resonance angle. The incidence position of each of the parallel reflected lights B group 240 is the surface position of the metal film 320 right beneath the corresponding measuring cell (one of measuring cells of m rows and n columns).
300 is a sensor which is a glass plate 310 having a metal film 320 vapor-deposited on the bottom surface. The sensor 300 is mounted on the upper surface of prism 410 via a matching plate not shown in
From the metal film 320 right beneath the measuring cells 330, on which the parallel reflected lights B group 240 is incident, there is radiated, at a reflection angle θb, a reflected lights C group 340 (each sample in each measuring cell gives a different angle of resonance owing to surface plasmon resonance phenomenon and, as a result, different reflected lights C are radiated from different samples). The reflected lights C group 340 passes through the prism 410 and then through a polarizer 420 b, is incident simultaneously on each corresponding position of the photodiode array detectors group 510 (of m rows and n columns) in the detector 500. The photodiode array detectors group 510 detects each SPR signal image generated by each measuring cell (of m rows and n columns) of the sensor 300, as an independent light spot.
Each translucent film used in the first optical system is preferred to convert 90.00 to 99.99% of the luminous energy of incident light into a transmitted light and 0.01 to 10.00% of the luminous energy into a reflected light, and is more preferred to convert 98 to 99% of the luminous energy of incident light into a transmitted light and 1 to 2% of the luminous energy into a reflected light. When the proportions of the transmitted light and the reflected light are in the above ranges, the luminous energy of the transmitted light (laser beam) incident on the last splitting unit Am is secured at a sufficient level. As a result, there are obtained, in all the splitting units, reflected lights each having a luminous energy necessary for the measurement of surface plasmon resonance phenomenon.
For the same reason as mentioned above, each translucent film used in the second optical system is preferred to convert 90.00 to 99.99% of the luminous energy of incident light into a transmitted light and 0.01 to 10.00% of the luminous energy into a reflected light, and is more preferred to convert 98 to 99% of the luminous energy of incident light into a transmitted light and 1 to 2% of the luminous energy into a reflected light.
As shown in
There is shown below an Example of 25-channel simultaneous analysis using the multi-channel surface plasmon resonance phenomenon measuring equipment of the present invention.
There were produced an optical system for multi-channel SPR, comprising a laser beam source, two multi-beam splitters, an angle-fixed trapezoidal prism, 25 rectangular photodiodes of 3×2 mm and an electric circuit, and a measurement soft ware; and a 25-channel sensor was evaluated.
The multi-beam splitters were produced as follows. That is, 7 glass plates (BK 7) of 50 mm×50 mm×1.77 mm (thickness) were prepared. On one side of each glass plate was formed a dielectric multi-layered film for adjustment of light transmittance. The formation of dielectric multi-layered film was carried out by conducting vacuum deposition using a substance containing titanium (a dielectric), as a source, and one side of each glass plate as a target, to apply multi-AR coating on the surface of glass plate. In this way, there were produced 7 light-reflecting mirrors showing a transmittance of 99% and a reflectivity of 1% to a light of 680 nm wavelength. Then, the light-reflecting mirrors were laminated to each other (the coating side of one mirror was directed upward and laminated to the non-coated side of other mirror by using a photo-curing adhesive [NOA 55 (registered trade name), a product of NOLAND Co., refractive index=1.52]), to produce a light-reflecting mirror laminate. From this light-reflecting mirror laminate were cut out splitting units.
The cut end faces of each splitting unit was polished at a profile irregularity of ⅛λ in order to obtain an increased forward movability of beam, whereby splitting units A and splitting units B were obtained.
The splitting units A and the splitting units B were arranged appropriately to produce a multi-beam splitter for laser beam, for 25-channel SPR simultaneous measurement. In the multi-beam splitter, the m and n both shown in
Then, the structure of the 25-channel sensor for SPR, used in the present Example is shown in
In the measurement of SPR, at first, the sensor was set at a predetermined position of the prism; then, samples of different glucose concentrations ranging from 1% to 10% were placed in 21 cells in order each in an amount of 5 μl using a pipette. In the remaining 4 cells were placed pure water for reference and, as standard solutions of given glucose concentrations, aqueous glucose (1, 3 and 5%) solutions, each in the same amount. Upon pushing the measurement button of the present measurement equipment, simultaneous SPR measurement was started immediately. One minute later, there was displayed, on the screen of computer, the measurement result for each channel, of the difference between the SPR intensity of water and the SPR intensity of sample to be measured, in relation to the concentration of glucose. Part of the display and the relation between SPR intensity and concentration are shown in Table 1 and
In order to clarify the applicability of the present invention to immunity measurement, there was investigated a 25-channel immunity sensor based on a IgG-antiIgG reaction. Measurement was conducted in the same manner as in Example 1 except that immunoglobulin IgG was used in place of glucose. As a result, the same result as in glucose was obtained and it has become clear that the present invention is applicable also to immunity measurement.
Therefore, the present invention has enabled palm-size, on-site, real time, multi-channel simultaneous analysis which has heretofore been impossible in conventional SPR measurement, and has made a new progress in SPR measurement field which is diversified.
In the above Examples, investigation was made with 25 channels which are small in channel number, from the standpoint of proving the effect of the present invention. However, since, in the present invention, the sensor can be made denser under the conditions that the incident lights cause no interference with each other, the sensor can be easily allowed to have more channels by preparing a microchip-like sensor with a micro-processing technique. For example, even a palm-size, 96-hole, novel ELISA equivalent can be produced easily. Further, when the sensor base plate limit of 30 mm×30 mm is exceeded under the requirement of palm size, more channels can be realized by making larger the base plate. Thus, the present invention has flexibility for the increase in number of channels. As a result, future utilization of the novel chemical analysis equipment based on the present invention is promising in fields wherein simultaneous analysis of a number of components has been needed, such as environment, agricultural chemical, food, drug, gene and the like. The present invention can apparently contribute to the efficient resolution of various problems arising from organic substances, and the present invention is considered to be very effective in the safety of human beings and the grassroots development of bioscience in the 21st century, an age in which reliability is established based on versatile information.